Fibroblast monolayers were established by explant culture of dermis from patients with PSS and from normal individuals. Prior to explanting, each of the biopsies was split into three fragments, so that monolayers were derived from a full-thickness portion of dermis (including epidermis and subcutis), an upper portion of dermis (including epidermis), and a lower portion of dermis (including subcutis). Collagen accumulation in the culture medium was measured as 3H-proline incorporation into CSP and as labeled hydroxyproline. Cultures from the patients with PSS accumulated significantly more CSP and labeled hydroxyproline than did normal cultures when monolayers derived from corresponding portions of the biopsies were lower dermal (PSS vs. NL, p less than 0.001). In addition, cultures from lower PSS dermis accumlated more collagen than those from upper PSS dermis (P less than 0.05), but this was not true for normal cultures (NL lower vs. NL upper, p greater than 0.05). When cultures were supplemented with additional ascorbic acid, both PSS and NL cultures showed an increase in accumlation of CSP and hydroxyproline. The increase in CSP was greater in PSS than in NL cultures. Data from several monolayers, followed in culture for as many as 15 generations, show that the difference between PSS and NL cultures can be propagated. These data show that scleroderma cultures consistently accumulate more collagen than do normal cultures.